Dissolving sodium hydrosulfide in drinking water is not a good source of hydrogen sulfide for animal studies

Hydrogen sulfide (H2S) has many physiological and pathological roles in the human body. Sodium hydrosulfide (NaHS) is widely used as a pharmacological tool for assessing H2S effects in biological experiments. Although H2S loss from NaHS solution is a matter of minutes, some animal studies use NaHS in solution as an H2S-donating compound in drinking water. This study addresses whether 30 μM NaHS in drinking water prepared in rat/mouse water bottles remains stable for at least 12–24 h, as presumed by some authors. NaHS solutions (30 μM) were prepared in drinking water and immediately transferred to rat/mice water bottles. Samples were obtained from the tip of water bottles and from inside of the bottles at 0, 1, 2, 3, 4, 5, 6, 12, and 24 h for sulfide measurement using the methylene blue method. Furthermore, NaHS (30 μM) was administered to male and female rats for two weeks, and serum sulfide concentrations were measured every other day in the first week and at the end of the second week. NaHS solution was unstable in the samples obtained from the tip of water bottles; it declined by 72% and 75% after 12 and 24 h, respectively. In the samples obtained from the inside of the water bottles, the decline in the NaHS was not significant until 2 h; however, it decreased by 47% and 72% after 12 and 24 h, respectively. NaHS administration did not affect serum sulfide levels in male and female rats. In conclusion, NaHS solution prepared in drinking water can not be used for H2S donation as the solution is unstable. This route of administration exposes animals to variable and lower-than-expected amounts of NaHS.


Ethical approval
All experiments of the current study were affirmed by the published guidelines for the care and use of laboratory animals in Iran 31 .All experiments of the current study were also reported following ARRIVE guidelines 32 .The ethics committee of the Research Institute for Endocrine Sciences, affiliated with the Shahid Beheshti University of Medical Sciences, confirmed and approved all experimental procedures of the current study.

Preparation of NaHS solutions
NaHS solutions (30 μM) was prepared in drinking water and immediately transferred to rat/mice water bottle.This concentration was chosen based on many publications that use NaHS as an H 2 S source in their studies; please see the discussion section.NaHS is a hydrated molecule and may have variable numbers of waters of hydration (i.e., NaHS•xH 2 O); according to the manufacturer, the percent of NaHS used in our study was 70.7% (i.e., NaHS•1.3H 2 O) and this value was considered in our calculations, where we used a molecular weight of 56.06 g/mol, which is for anhydrous NaHS.Waters of hydration (also called waters of crystallization) are water molecules integral to the crystal structure 33 .Hydrates have different physical and thermodynamic properties compared to anhydrates 34 .
The solvent's pH and temperature were measured before adding the NaHS to the drinking water.NaHS solution was immediately transferred to the water bottles of rats/mice in the animal cages.Samples were obtained from the tip of water bottles and from inside of the bottles at 0, 1, 2, 3, 4, 5, 6, 12, and 24 h for sulfide measurement.Sulfide measurement was done immediately after each sampling.We obtained samples from the tip of the bottles as some studies have stated that the small aperture of water bottles minimizes H 2 S evaporation 15,19 .This issue seems to be true for the solution inside the bottle.However, this is not the case for solution in the tip of water bottles with a higher evaporation rate and auto-oxidation; indeed, animal drinks this water first.

Experiments in rats
In this study male and female Wistar rats were used.Rats were housed in polypropylene cages (2-3 rats/cage) under standard conditions (temperature 21-26 °C and humidity 32-40%) of 12-h light (7 am to 7 pm) and 12-h dark (7 pm to 7 am).They had free access to tap water and the regular chow diet (Khorak Dam Pars, Co., Tehran, Iran).Age-matched (6-month old) female (n = 10, body weight: 190-230 g) and male (n = 10, body weight: 320-370 g) Wistar rats were randomly assigned to control and NaHS (30 μM)-treated groups (n = 5/ group).We used the KISS (Keep It Simple, Stupid) approach to determine sample size, which combines past experience with power analysis 35 .We first performed a pilot study with 3 rats and determined the mean total serum sulfide levels and standard deviation (8.1 ± 0.81 μM).Then, considering 80% power and assuming a twosided 5% significance level, we determined our provisional sample size (n = 5 based on previous literature) that corresponded to a standardized effect size of 2.02 from predefined values presented by Festing for sample size calculation in laboratory animals 35 .After multiplying this value by SD (2.02 × 0.81), the predicted detectable effect size (1.6 μM) was 20%, which is acceptable.It means that n = 5/each group is sufficient to detect a mean change of 20% between groups.Randomization of rats to control and NaSH-treated groups was done using the random function of the Excel software 36 (Supplementary Fig. 1).Blinding was done at the outcome level, and the researcher who performed biochemical measurements was blinded to the groups.
NaHS groups of both sexes were treated with 30 μM of NaHS solution prepared in drinking water for 2 weeks; fresh solutions were provided every 24 h, at which time body weights were measured.Blood samples were obtained from the tail tips of all rats under isoflurane anesthesia every other day in the first week and at the end of the second week.Blood samples were centrifuged at 3000 g for 10 min, and sera were separated and maintained at -80 °C for subsequent measurement of serum urea, creatinine (Cr), and total sulfide.Serum urea was measured by the enzymatic urease method and serum Cr by the photometric Jaffe method using commercially available kits (Man Company, Tehran, Iran) and an auto-analyzer (Selectra E, serial number 0-2124, Netherlands).Intra-and inter-assay coefficient of variation for both urea and Cr were < 2.5%.

Total sulfide measurement
The methylene blue (MB) method was used to measure total sulfide in NaHS-containing drinking water and serum; MB is the most commonly used method for sulfide measurement in stock solutions and biological samples 11,37 .The MB method is useful for estimating total sulfide pools 38 and measures inorganic sulfide present as H 2 S, HS -, and S 2 in the aqueous phase 39 .In this method, sulfur is precipitated as zinc sulfide (ZnS) in the presence of zinc acetate 11,38 .Precipitation by zinc acetate is the most widely used technique for separating sulfide from other chromophores 11 .ZnS is redissolved under highly acidic conditions using HCl 11 .The sulfide reacts with DMPD in ferric chloride (Fe 3+ acts as an oxidizing agent) catalyzed reaction with 1:2 stoichiometric ratio to give the MB dye, which is detected spectrophotometrically at 670 nm 40,41 .The detection limit of the MB method is around 1 µM 11 .
In the current study, 100 μL of each sample (solution or serum) was added to a test tube; then, 200 μL of zinc acetate (1% w/v in distilled water), 100 μL of DMPD (20 mM in HCl 7.2 M), and 133 μL of FeCl 3 (30 mM in HCl 1.2 M) were added.The mixture was incubated in a dark environment at 37ºC for 30 min.The solution was centrifuged at 10,000 g for 10 min, and the absorbance of the supernatant was read at 670 nm using a microplate reader (BioTek, MQX2000R2, Winooski, VT, USA).A NaHS (0-100 μM) calibration curve in ddH2O was used to determine sulfide concentrations (Supplementary Fig. 2).All solutions used for measurement were prepared freshly.Intra-and inter-assay coefficients of variation of sulfide measurement were 2.8% and 3.4%, respectively.Using the spiked samples method, we also determined the total sulfide recovered for NaSH-containing drinking water and serum samples 42 .Recovery values for NaSH-containing drinking water and serum samples were 91 ± 1.1% (n = 6) and 93 ± 2.4% (n = 6), respectively.

Statistical analyses
GraphPad Prism version 8.0.2 for Windows (GraphPad Software, San Diego, California USA, www.graph pad.com) was used for statistical analyses.The temperature and pH of drinking water before and after adding NaHS were compared using a paired t-test.Loss of H 2 S from NaHS-containing solutions was calculated as the percent decrease from baseline absorbance, and to assess whether this loss is statistically significant, one-way repeated measure ANOVA followed by the Dunnett multiple comparison test was used.Two-way mixed (between-within) ANOVA with the Bonferroni posthoc test was used for comparing body weight, serum urea, serum Cr, and serum total sulfide between control and NaHS-treated rats over time in each sex.Two-sided P-values < 0.05 were considered statistically significant.

Stability of NaHS solution
The pH of the drinking water was 7.60 ± 0.01 before the addition of the NaHS and 7.71 ± 0.03 (n = 13, p = 0.0029) after adding NaHS.The drinking water temperature was 26.5 ± 0.2, which decreased to 26.2 ± 0.2 (n = 13, p = 0.0128) after adding NaHS.30 μM solutions of NaHS were prepared in drinking water and maintained in the water bottles.The NaHS solution was unstable, and its concentration declined with time.When samples were obtained from the tip of the water bottles, a large decrease (68.0%) was observed during the first hour, and the amount of NaHS in the solution declined by 72% and 75% after 12 and 24 h, respectively.In the samples obtained from the inside of the water bottles, the decline in the NaHS was not significant until 2 h; however, it decreased by 47% and 72% after 12 and 24 h, respectively.These data indicate that regardless of the sampling location, the percent of NaHS in a 30 μM solution prepared in drinking water is decreased to about one-fourth of the initial value after 24 h (Fig. 1).

Changes in NaHS concentration of drinking water
The concentration of NaHS in freshly prepared 30 μM solutions was 30.3 ± 0.4 μM (range: 28.7-31.9μM, n = 12).However, it declined to low values (mean: 3.0 ± 0.6 μM) after 24 h.As shown in Fig. 2, rats were not exposed to a stable concentration of NaHS during the study.; however, NaHS administration did not affect body weight (Fig. 3).Body weight of male rats increased significantly over time (from 338.6 ± 8.3 to 352.4 ± 6.0 in control and from 352.4 ± 5.9 to 363.2 ± 4.3 g in NaHS-treated rats); however, NaHS administration did not affect body weight (Fig. 3).Serum urea and Cr concentrations were comparable in control and NaSH-treated rats throughout the study.In addition, NaSH treatment did not affect serum urea and Cr concentrations (Table 1).

Discussion
The main result of this study is that NaHS-containing drinking water is not stable, and only about one-fourth of the baseline total sulfide was recovered after 24 h following sampling from the tip and inside of rat/mice water bottles.In addition, due to H 2 S loss from the NaHS solution, rats were not exposed to a stable concentration of NaHS, and NaHS administration in drinking water did not affect body weight, serum urea and Cr, and serum total sulfide levels.
In this study, the rate of H 2 S loss was about 3% per hour of 30 μM NaHS solutions prepared in drinking water.Time-dependent decrease in sulfide concentration has been reported to be 7% per hour in buffer solutions (sodium sulfide, 100 μM in 10 mM PBS, pH = 7.4) over 8 h 11 .We previously, in defense of intraperitoneal injection of NaHS, reported that the sulfide loss rate from NaHS-containing drinking water in a 54 μM solution was  4%/hour for the second 12 h after its preparation) 8 .The steady loss of H 2 S from NaHS solution has been emphasized in earlier works 43 and is mainly done via volatilization and oxidation.Even without bubbling, sulfide loss from stock solutions via H 2 S volatilization is fast 11 .It has been shown that during dilution of stock solution, which took about 30-60 s, about 5-10% of H 2 S is lost by evaporation 6 .To avoid H 2 S evaporation from the solutions, researchers have adopted some measures, including gentle mixing of solutions 12 , covering the stock solutions with parafilm 6 , and minimizing exposure of the solutions to air, as the rate of H 2 S evaporation depends on the air-liquid interface 13 .Spontaneous oxidation Table 1.Changes in serum urea and creatinine (Cr) (mg/dL) of male and female rats in control and NaSHtreated (30 μM) (NaSH) groups at different time points.Data are mean ± SEM and were compared using twoway mixed (between-within) ANOVA with the Bonferroni posthoc test.n = 5/group in each sex.www.nature.com/scientificreports/ of H 2 S is primarily due to transition metal ions, particularly ferric iron, which exists as an impurity in water 13 .Oxidation of H 2 S causes the formation of polysulfides (sulfur atoms connected with covalent bonds) 11 .To avoid its oxidation, H 2 S-containing solutions are prepared in deoxygenated solvents 44,45 following purging solutions with argon or nitrogen for 20-30 min to ensure deoxygenation 11,12,37,[44][45][46] .Diethylenetriamine-pentaacetic-acid (DTPA), a metal chelator (10 -4 M), prevents auto-oxidation of HS -in aerobic solutions, which in the absence of DTPA is about 50% in about 3 h at 25ºC 37,47 .In addition, solutions should be kept in the dark on ice as 1e -sulfide oxidation is catalyzed by UV light 11 .
As shown in Fig. 5, when NaHS is dissolved in water, it ionizes to Na + and HS -6 ; this dissociation depends on pK 1 of the reaction, which is temperature-dependent: pK 1 = 3.122 + 1132/T, where T is within 5 to 30º C and used in degrees of Kelvin (K) and K = ºC + 273.15 48.pK 2 of HS -is high (pK 2 = 19), thus, it does not yield S 2-or minimal amounts of S 2-produced at pH < 9 6,49 .Instead, HS − acts as a base and accepts H + from the H 2 O molecule, which acts as an acid and is converted to H 2 S and OH − .Despite evidence that NaHS solutions are not stable, some animal studies used NaHS solutions in drinking water as an H 2 S-donating compound [15][16][17][18][19][20][21][22][23][24][25][26] , with the duration of intervention ranging from 1 to 21 weeks (Table 2).These studies refreshed NaHS solutions every 12 15,17,18,24,25 or 24 [19][20][21][22][23] h.Our results indicate that due to H 2 S loss from the NaHS solution, rats are not exposed to a stable concentration of the drug, and a large fluctuation in the content of NaHS in the drinking water of rats occurs over 12 or 24 h (see Fig. 2).Two of these studies reported that H 2 S content in water was stable for over 24 h 22 or only 2-3% H 2 S loss was observed over 12 h 15 , but they did not provide supporting data or details of the measurement.Two studies stated that the small aperture of water bottles minimizes H 2 S evaporation 15,19 .However, our results showed that this only retards H 2 S loss from the inside of the water bottle for 2 h, not 12-24 h.In two studies, it has been stated that we presumed that NaHS content in the drinking water was not changed as we did not see changes in water coloring; thus, oxidation of  www.nature.com/scientificreports/H 2 S due to air is not significant 19,20 .It is a surprise that such a subjective method is used for assessing the stability of NaHS in water instead of measuring its concentration over time.H 2 S loss from NaHS solutions is pH-and temperature-dependent.Dissolving NaHS in water produces an alkaline solution 50 , as observed in our study.The production of dissolved H 2 S gas, following the dissolution of NaHS in water, is pH-dependent 6 .The lower the pH solution, the larger the fraction of NaHS in the form of the H2S gas molecules, and the more sulfide is lost from aqueous solutions 11 .None of these studies reported the pH of the drinking water used as the solvent for NaHS.According to WHO guidelines that most countries have adopted, the pH value of drinking water should be in the range of 6.5-8.5 51 .The rate of spontaneous H 2 S oxidation increased by about tenfold over this pH range 13 .Dissolving NaHS in the water with this pH range yields the concentration of dissolved H 2 S gas to be between 1 to 22.5 μM, highlighting the importance of controlling water pH before dissolving the NaHS.In addition, the temperature range reported in the studies mentioned above (18-26 °C) causes about 10% changes in concentration of dissolved H 2 S gas in the solution, as a change in the temperature changes pK 1 and a slight change in pK 1 has a substantial effect on the fraction of dissolved H 2 S gas 48 .Adding to this issue is the long duration of some studies (5 months) 22 , during which more variability in temperature is expected to occur.
All studies except one 21 used 30 μM of NaHS solutions in drinking water.To explain the dose used (i.e., 30 μM), some authors stated that NaHS in the aqueous phase produces exactly equal concentrations of H 2 S gas, and since the physiological range of H 2 S is between 10 and 100 μM, this dose is in the physiological range 15,16 .It has also been explained that 30 μM NaHS keeps plasma H 2 S levels in the physiological range, i.e., 5-300 μM 19,20 .Let us consider a concentration of 30 μM of NaHS in water (pH = 7.0, and T = 20 °C), which had been used in some studies to investigate the effects of H 2 S. We can calculate the concentration of dissolved H 2 S gas to be 14.7 μM, which is about 50% of the initial concentration of NaHS.This value is similar to previous calculations by others under the same conditions 13,48 .
In our study, NaHS administration did not change body weight; this finding is in line with other studies in male mice 22,23 and male rats 18 ; however, two studies reported that NaSH restored reduced body weight in nephrectomized rats 24,26 and other studies did not report an effect of NaSH administration on body weight [15][16][17][19][20][21]25 . In additin, in our study, NaSH administration did not affect serum urea and Cr, a finding that aligns with another report 25 .
This study found that NaHS administration in drinking water for 2 weeks did not affect serum total sulfide concentrations in male and female rats.This finding is in line with results reported by Sen et al. 16 that 8-week treatment with 30 μM NaHS in drinking water did not affect plasma sulfide levels in control rats; however, they reported that this intervention restored decreased plasma H 2 S levels in nephrectomized mice.Lee et al. also reported that 5 months of treatment with 30 μM NaHS in drinking water increased plasma free sulfide levels by about 26% in aged mice 22 .Other studies have not reported circulating sulfide changes following NaHS intervention in drinking water.
Seven studies have reported that they used NaHS from Sigma 15,16,[19][20][21][22][23] but did not provide more details about water of hydration, and five studies did not mention the source of NaHS used for their preparations 17,18,[24][25][26] .NaHS is a hydrated molecule and may have variable waters of hydration, affecting the amount of NaHS needed for preparing a given molar concentration of the solution.For example, in our study, it was as NaHS•1.3H 2 O. Therefore, the actual concentrations of NaHS in these studies may be lower than those reported.
"How could such a short-lived compound exert such sustained effects?" this question has been asked by Pozsgai et al. 21, who assessed the effects of NaHS on colitis in mice.These authors hoped that future studies would answer this question and speculated that the NaHS solution might contain more stable polysulfides besides H 2 S and bisulfides that mediated the NaHS effects 21 .Another possibility is that a very low concentration of NaHS remaining in the solution can provide beneficial effects.Indeed, Olson has provided evidence that the micromolar range of H 2 S in the blood is unphysiological, and it should be in the nanomolar range, or it does not exist in blood at all 13 .There is a possibility that H 2 S acts through protein sulfhydration, which is a reversible post-translational modification and affects the function, stability, and localization of the many proteins [52][53][54] .Indeed, about 10-25% of many liver proteins are sulfhydrated under physiological conditions 53 .Two studies acknowledged the fast decay of NaHS 19,23, but surprisingly, they stated that "we controlled the concentration of NaHS in the drinking water by changing the drinking water every day" 23 .One study surprisingly claims that "NaHS is a standard H 2 S donor which is usually used in clinic instead of H 2 S itself " 18 .
According to the discussion above indicating that NaHS is lost from solution by volatilization, oxidation, and photodecomposition, some suggestions have been provided for minimizing H 2 S loss from solution.First, H 2 S evaporation depends on the air-liquid interface 13 and pH of the solution 11 ; thus, for minimizing loss from volatilization, the size of the aperture of water bottles can be minimized as much as possible, as suggested previously 15,19 and pH of water can be adjusted at the upper acceptable range (i.e., 6.5-8.5 51 ) to minimize loss from volatilization 11 .Second, spontaneous oxidation of H 2 S is due to exposure to oxygen and the presence of transition metal ions in drinking water 13 , therefore deoxygenating drinking water using argon or nitrogen 44,45 and using a metal chelator 37,47 can decrease sulfide oxidation.Third, to prevent the photodecomposition of H 2 S, water bottles can be wrapped in aluminum foils; this practice is also applied to light-sensitive materials, such as streptozotocin 55 .Finally, inorganic sulfide salts (NaHS, Na 2 S, and CaS) can be gavaged instead of dissolving in drinking water as has been reported previously [56][57][58] ; it has been shown that following gavage of radioactive sodium sulfide to rats, it is well absorbed and distributed in almost all tissues 59 .Currently, most studies administer inorganic sulfide salts via the intraperitoneal route; however, this route is minimally used in the clinic 60 .On the other hand, the oral route is the most common and preferred route of drug administration in humans 61 .Thus, we suggest oral gavage for assessing the effects of H 2 S donors in rodents.
As a limitation, we measured sulfide levels in aqueous solutions and serum using the MB method.Methods for measurement of sulfide include iodometric, spectrophotometric, electrochemical (potentiometric, galvanic, coulometric, and amperometric), and chromatographic (gas chromatography and HPLC), of which spectrophotometric MB method is the most common approach 62 .A limitation of the MB method for measuring H 2 S levels in biological samples is that it measures all sulfur species rather than free H 2 S 63 because it is done under acidic conditions, which extracts sulfur from biological sources 64 .However, according to the American Public Health Association, MB is the standard method for measuring aqueous sulfide 65 .Therefore, this limitation does not affect our main result on the instability of the NaHS-containing solution.In addition, our study's sulfide measurement recovery was 91% and 93% for NaHS-containing water and serum samples, respectively.These values are in line with the previously reported range (77-92) 66 and indicate reasonable accuracy of the assay 42 .As a strength, we used both sexes of rats according to the recommendation of the National Institutes of Health (NIH) to avoid over-reliance on male-only animal research in preclinical studies 67 and include both sexes when possible 68 .Others have emphasized this issue [69][70][71] .
In conclusion, the findings of this study indicate that NaHS solution prepared in drinking water cannot be used for H 2 S donation as the solution is not stable.This route of administration exposes animals to variable and lower-than-expected amounts of NaHS; data derived, therefore, may not be translated to humans.

Figure 1 .
Figure 1.Stability of NaHS solution (30 μM) in drinking water maintained in rat/mice water bottle.After the solution was prepared, samples were obtained from the tips of the water bottles and the inside.Data are mean ± SE (n = 6/group).* and #, P < 0.05 compared to time 0. Picture of water bottle indicating the tip (with its aperture) and body.The volume of the tip is about 740 μL.

Figure 2 .
Figure 2. Changes in NaHS concentrations of drinking water to which rats were exposed during the study.

Figure 3 .
Figure 3. Changes in body weight following administration of NaHS (30 μM) in female and male rats.Data are mean ± SEM and were compared using two-way mixed (between-within) ANOVA with the Bonferroni posthoc test.n = 5/group in each sex.

Figure 4 .
Figure 4. Changes in serum total sulfide concentrations following administration of NaHS (30 μM) in female and male rats.Data are mean ± SEM and were compared using two-way mixed (between-within) ANOVA with the Bonferroni posthoc test.n = 5/group in each sex.

Table 2 .
Summary of studies that administrated NaHS in drinking water in rodents.NR not reported.